ABSTRACT Progressive Multifocal Leukoencephalopathy (PML) is a life-threatening demyelinating brain disease in immune-compromised individuals caused by the JC polyomavirus (JCPyV), a ubiquitous human pathogen. No anti-JCPyV agents are available. Among the expanding compendium of PML-associated biologics, natalizumab has the highest incidence (1-2%) and is an ominous complication for multiple sclerosis (MS) patients who otherwise benefit from dramatic reductions in relapses using this immunomodulatory agent. Drug withdrawal, the only therapeutic option for PML, is often complicated by a high-mortality cerebral inflammatory reaction. Moreover, three non-PML JCPyV-CNS diseases have recently been described. Lack of a tractable animal model of polyomavirus-induced CNS disease is a well-recognized bottleneck to elucidating PML pathogenesis and the immunological mechanisms that control JCPyV (both essential for identifying PML risk factors), and in vivo evaluation of antiviral agents that inhibit polyomavirus replication in tissue culture. Using mouse polyomavirus (MuPyV), we developed a natural virus-host model of polyomavirus-associated demyelination. In work supported by the parent R01, we found a sizeable MuPyV-specific, brain resident- memory CD8 T cell (bTRM) population. TRM are disseminated throughout the body as non-recirculating cells, where they provide frontline defense against reinfection. Our understanding of TRM biology comes largely from analysis of these cells in mucosal barriers; far less is understood about TRM in nonmucosal sites of infection, particularly those with large numbers of nonrenewable cells (e.g., CNS). We discovered a critical connection between CD4 T cells and differentiation of functional MuPyV-specific CD8 bTRM. Our preliminary data provide strong scientific premise for our central hypothesis that IL-21 is the ?help? proffered by CD4 T cells for generating MuPyV-specific CD8 bTRM. CD4 T cell insufficiency and IL-21 signaling-deficiency each resulted in loss of antiviral CD8 T cells expressing a TRM phenotype and inability to survive in the brain without resupply from the circulation. Gene expression pathway analyses of CD4 T cell-unhelped antiviral CD8 T cells during persistent MuPyV encephalitis revealed dysregulation of IL-21 and Notch signaling pathways, and their potential convergence via STAT3. MuPyV-specific CD8 bTRM express functional Notch receptors, whose activation is diminished in the absence of CD4 T cell help. Notch receptors have recently been reported to support establishment of tissue-resident memory CD8 T cells. For Specific Aim 1, we hypothesize that IL-21 is a major component of the help provided by CD4 T cells to guide differentiation of brain-infiltrating CD8 T cells into TRM. For Specific Aim 2, we hypothesize that IL-21 and Notch signaling cooperate via STAT3 activation to generate CD8 bTRM. If these hypotheses prove correct, they will provide essential insights into the mechanisms involved in maintaining and sustaining immunologic control of polyomavirus CNS infection.